The ultimate objective of the research proposed here is to delineate the regulation of gene expression for protein N-glycosylation during the hormonally-modulated growth and differentiation of the mammary gland. The focus of the investigation is to purify and characterize the GDP-Mannose- requiring mannosyltransferases of the dolichol cycle int he rat mammary gland and develop molecular reagents, viz, antibodies and cDNAs for the same. These studies are being proposed in the context of earlier studies from the P.I.s lab showing that a number of enzymes of protein N- glycosylation are modulated during the ontogeny of the mammary gland; the combined action of insulin, prolactin and hydrocortisone appears to regulate their expression over basal levels. Our working hypothesis postulates that the dolichol-linked assembly and polypeptide-linked maturation of the oligosaccharide precursor for N-glycosylation are coordinately upregulated by the synergistic action of the above hormones during the lactogenic differentiation of the rat mammary gland. Asparagine-linked glycoproteins comprise the largest class of glycoproteins and are involved in a myriad of phenomena that are fundamental to biological recognition. Alterations in glycoprotein metabolism are associated with numerous pathologies and host-parasite interaction. A concert of seventeen glycosyltransferases and two glycosidases is common to the assembly of all N-linked glycoproteins. Among these five GDP-Mannose requiring mannosyltransferases constitute a family of enzymes. Using a novel, tripartite strategy, one of these enzymes has already been purified and characterized in the P.I's lab. The proposed study will extend the same overall approach to procure the other enzymes. A specific goal is to identify a potential "mannosyl motif" within the active site of these enzymes. An experimental strategy is outlined, analogous to the active site-SH tagging methodology developed in the P.I.'s laboratory to obtain catalytic polypeptides of the enzymes for antibody production, should it be difficult to obtain homogenous enzymes. Standard PCR-based technologies will be employed to obtain the cDNAs for the enzymes. The availability of these reagents should open the door for future investigations on the regulation of protein N-glycosylation in animal systems. The mammary gland offers a unique model to the investigator for studying glycoprotein biosynthesis and regulation at the biochemical and molecular-biological level. It is intensely modulated by a variety of hormones for its growth and differentiation throughout the reproductive life of the female. It can potentially serve as an excellent bioreactor in transgenic animals to harvest large quantities of biomedically significant glycoproteins in its secretion, i.e., milk. Preliminary successes with the secretion of alpha- antitrypsin, tissue plasminogen activator, and blood clotting factors appear very promising.